HIV protease inhibitors, such as, ritonavir and saquinavir, are substrates for xenobiotic efflux pumps, e.g., P-glycoprotein and Mrp2 and thus penetrate the blood-brain barrier poorly. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice in vivo to validate signaling-based changes in blood-brain barrier transporter function. Our recent studies have focused on identifying signals that modify pump activity in the barrier. These in vitro and in vivo experiments with animal models suggest multiple strategies for specifically modifying this barrier, but also potentially important complications of polypharmacy. Strategies for improving drug delivery of drugs that are P-glycoprotein substrates include: activating VEGF signaling through src kinase and specifically activating protein kinase C (PKC) isoform beta1. Both strategies cause rapid and reversible loss of P-glycoprotein transport activity in isolated brain capillaries. In rats in vivo, such signaling increases brain uptake of drugs that are P-glycoprotein substrates. Complications related to polypharmacy involve increased expression of multiple blood-brain barrier drug efflux pumps after exposure to therapeutic drugs, dietary constituents and environmental toxicants that specifically activate the nuclear receptors, PXR, CAR or AhR. Once activated by ligand, these receptors move into the nucleus and target the promoter regions of xenobiotic metabolizing enzymes and efflux transporters. In brain capillaries in vitro or in vivo, exposure to receptor ligands more than doubles expression and transport activity of P-glycoprotein, Mrp2 and BCRP. These findings raise the possibility of further tightening of the blood-brain barrier to many therapeutic drugs in patients undergoing polypharmacy.